Synthesis of new tetrahydropyridinylidene ammonium salts and their antiprotozoal potency

Article abstract of DOI:10.1007/s00706-015-1503-y

Several new tetrahydropyridinylidene ammonium salts were prepared by selective reduction. They were characterized using UV-Vis spectroscopy, FT-IR spectroscopy, and HRMS. Their structure was established by NMR spectroscopy and a single X-ray structure ana

Full text of DOI:10.1007/s00706-015-1503-y

Monatsh Chem
DOI 10.1007/s00706-015-1503-y
Synthesis of new tetrahydropyridinylidene ammonium salts
and their antiprotozoal potency
1
1
3
2
•
Werner Seebacher
4
•
Johanna Faist
4
•
Ferdinand Belaj
1
•
Robert Saf
Marcel Kaiser
•
Reto Brun
•
Robert Weis
Received: 29 April 2015 / Accepted: 27 May 2015
Ó Springer-Verlag Wien 2015
Abstract Several new tetrahydropyridinylidene ammo-
nium salts were prepared by selective reduction. They were
characterized using UV–Vis spectroscopy, FT-IR spec-
troscopy, and HRMS. Their structure was established by
NMR spectroscopy and a single X-ray structure analysis.
One compound shows a distinct antiplasmodial potency
Introduction
According to the latest estimates, malaria mortality rates
were reduced by about 42 % globally and by 49 % in the
WHO African Region between 2000 and 2012. But parasite
resistance to artemisinin—the core compound in the
world’s most effective antimalarial medicines—and mos-
quito resistance to insecticides remain major concerns. If
not addressed with appropriate urgency, they could threa-
ten the remarkable progress made since 2000 [1]. So the
development of new and effective antimalarial drugs is of
urgent need.
(
IC50 = 0.34 lL) against the multiresistant K -strain of
1
Plasmodium
IC50 = 199.8 lL) against L-6 cells (rat skeletal
myoblasts).
falciparum
and
low
cytotoxicity
(
Graphical abstract
Human African trypanosomiasis, which is fatal if left
untreated, affects rural populations in sub-Saharan Africa.
Although the present number of cases (50,000–70,000)
seems negligible on a worldwide scale, the characteristics
and focal distribution of the disease can have a great
socioeconomic effect on affected villages. Diagnosis and
treatment are unsatisfactory and needs more research and
development [2]. Only five drugs are in use at the present,
which are not effective against all stages and strains of
trypanosomes. Melarsoprol, an organic arsenical, is the
only drug used to treat late stage Trypanosoma brucei
rhodesiense infection. Unfortunately, this drug induces an
extremely severe post-treatment reactive encephalopathy
Keywords Reductions Á X-ray structure determination Á
Heterocycles Á Drug research
(
PTRE) in up to 10 % of treated patients, half of whom
&
Werner Seebacher
we.seebacher@uni-graz.at
die from this complication [3]. Therefore, new try-
panocidal compounds with less side effects are of great
demand.
1
2
Institute of Pharmaceutical Sciences, University of Graz,
Graz, Austria
Several 4-aminopyridines showed antimalarial activity
[4–6]. We, therefore, prepared a series of new pyridinyli-
dene salts with differing 4-amino substitution and
investigated them for their activities against Plasmodium
falciparum and T. b. rhodesiense as well as for their
cytotoxicity.
Institute for Chemistry and Technology of Materials (ICTM),
University of Technology, Graz, Austria
3
4
Institute of Chemistry, University of Graz, Graz, Austria
Swiss Tropical and Public Health Institute Basel and
University of Basel, Basel, Switzerland
123

W. Seebacher et al.
1
3
Results and discussion
Chemistry
compounds 6. In C spectra, the signal of a quaternary carbon
for the thione group at 188 ppm is exchanged with a signal for
an olefinic carbon at 155 ppm. The crystal structure analysis
of 6a confirmed the compound as 4-(dimethylamino)-2,2-
dimethyl-2,3-dihydropyridinium iodide (Fig. 1) and is the
first structure determination of a protonated 4-amino-2,3-di-
hydropyridine. All atoms lie on general positions. By the
protonation of N1 a planarization of N4 (sum of the three
bond angles: 359.44° at N4, 358.7° at N1) is observed
according to the two following mesomeric structures which
are supported by the determined bond lengths (N1–C2
Our synthetic pathway started from mesityl oxide (1) and
thiocyanates of secondary amines 2a–2d reacting to
tetrahydropyridinethiones 3a–3d [7]. Alternatively, a
reaction of 4-isothiocyanato-4-methylpentan-2-one (4)
with secondary amine 5b yielded 3b [8]. Thiones 3a–3d
were transformed to the tetrahydropyridinylidene salts 6a–
6
d by a S-methylation/reduction procedure using deacti-
˚ ˚ ˚
1.473(2) A, N1–C6 1.317(2) A, C4–N4 1.3229(19) A, C2–C3
vated Raney nickel for the selective reduction process. To
vary the 4-amino substituents of 6, it was in some cases
easier to prepare thiones 3e–3j via reaction of compound 7
and the appropriate amine 5e–5j [9] followed by above-
mentioned steps to yield 6e–6j (Scheme 1).
˚
1.532(2) A, C4–C3 1.506(2) A, C4–C5 1.402(2) A, C5–C6
˚
˚
˚
1.377(2) A). In the packing (Fig. 2), the ion-pairs [N1–
˚
H1ÁÁÁI1 172(2)°, N1–H1ÁÁÁI1 3.5421(15) A] are arranged with
the iodides in tubes parallel to the a axis.
The preservation of the double bonds in compounds 6 was
1
verified by H NMR measurements. Instead of a singlet at
Antiprotozoal activity
5
.2 ppm for the olefinic proton in compounds 3, two doublets
with a coupling constant of approximately 6 Hz appear at 5.2
and 7.6 ppm for the olefinic protons at positions 5 and 6 in
All new tetrahydropyridinylidene salts 6a–6j were tested
against Plasmodium falciparum (NF54 strain) and
Scheme 1
1
23

Synthesis of new tetrahydropyridinylidene ammonium salts and their antiprotozoal potency
giving after aminolysis with aniline compound 10. A sub-
sequent Dimroth reaction leads to thione 11 which can be
converted by our S-methylation/reduction process to the
desired product 12 (Scheme 2).
This compound 12 was also tested for its antiprotozoal
activities. The result was a distinct increase of the
antiplasmodial activity into the submicromolar area
(IC50 = 0.36 lM), so the attachment of the phenyl ring
causes a 7-fold increase in activity (Table 1). Fortunately,
the toxicity remains nearly the same compared with 6c (6c:
IC50 = 192.4 lM; 12: IC50 = 199.8 lM), therefore, com-
pound 12 shows high selectivity (SI = 555). The activity
against the multiresistant strain Plasmodium falciparum K1
was even better, the IC50 was 0.34 lM.
Conclusion
The investigation of a series of tetrahydropyridinylidene
salts, which were produced by a selective S-methylation/
reduction process from their corresponding thiones
revealed, that the amino substitution in position 4 of such
compounds influences the antiprotozoal activity distinctly.
The 4-pyrrolidino-substituted compound showed highest
antiplasmodial activity which was 7-fold increased by an
attachment of a phenyl residue to the pyridine nitrogen.
From these results, further investigations seem to be
worthwhile.
Fig. 1 Stereoscopic ORTEP [10] plot of the asymmetric unit of 6a
showing the atomic numbering scheme. The probability ellipsoids are
drawn at the 50 % probability level. The H atoms are drawn with
arbitrary radii. The hydrogen bond is indicated by a dashed line
Experimental
Trypanosoma brucei rhodesiense as well as for their
cytotoxicity which was determined using L-6 cells. For
comparative reasons, some of the thiones 3a–3d were
tested too. The results are presented in Table 1.
Melting points were obtained on a digital melting point
apparatus Electrothermal IA 9200. IR spectra: infrared
spectrometer system 2000 FT (Perkin Elmer). UV/VIS:
Lambda 17 UV/VIS-spectrometer (Perkin Elmer) carried
The antiprotozoal potency of thiones 3a–3d is low, in
fact they are not active at all. Concerning the antiprotozoal
properties of the tetrahydropyridinylidene salts 6a–6j, the
promising antitrypanosomal potency of compounds 6i
out in CH OH solutions. NMR spectra: Varian Inova 400
3
(300 K) 5 mm tubes, spectra were acquired in DMSO-d6.
Chemical shifts were recorded in parts per million (ppm),
1
for H spectra the solvent signal at 2.49 ppm was used as
(
IC50 = 3.52 lM) and 6j (IC50 = 5.40 lM) is worth
1
3
mentioning. Furthermore, the selective reduction to
tetrahydropyridinylidene salts 6a–6d causes a distinct
increase of the antiplasmodial activity in case of substitu-
tion a, c, and d. The other compounds 6d–6j show
comparable potencies. The most active compound of this
series is N-(2,2-dimethyl-1,2,3,4-tetrahydropyridin-4-yli-
dene)pyrrolidiniumiodide (6d, IC50 = 2.53 lM) showing a
good selectivity (SI = IC50tox./IC50p.f. = 76). It was tested
against the multiresistant strain Plasmodium falciparum K1
standard and for C spectra the central peak of the DMSO
peak was used as the internal reference (39.7). Abbrevia-
tions: aromatic H, ArH; aromatic C, ArC, quaternary
aromatic C, ArC . Signal multiplicities are abbreviated as
q
follows: br, broad; d, doublet; m, multiplet; q, quartet; s,
singlet. Coupling constants (J) are reported in Hertz (Hz).
1
13
H and C resonances were assigned using H, H- and
1
1
1
13
H, C-correlation spectra. H and C resonances are
1
13
numbered as given in the formulae. The signals with an
asterisk are interchangeable. HR-MS: GCT-Premier,
Waters (EI, 70 eV), Micromass tofspec 3E spectrometer
(MALDI). Crystal structure analysis was performed on a
STOE four circle diffractometer. Materials: column chro-
matography (CC): silica gel (Merck, silica gel 60
(
resistant to chloroquine and pyrimethamine) showing an
even higer activity (IC50 = 1.73 lM). This was the reason
for a further derivatization of compound 6c. To insert an
aromatic residue attached to the ring nitrogen, we started
from thiopyrane thione 8 which was S-methylated to 9
123

W. Seebacher et al.
Fig. 2 Stereoscopic ORTEP [10] plot of the packing of 6a. The atoms are drawn with arbitrary radii, the hydrogen bonds are indicated by dashed
lines
(
0.063–0.200 mm); thin-layer chromatography (TLC):
TLC plates (Merck, silica gel 60 F254 0.2 mm,
00 9 200 mm); the substances were detected in UV light
as light brownish prisms. M.p.: 225 °C, corresponds well
with the reported melting point: 220 °C [11].
2
5
2
,6-Dihydro-6,6-dimethyl-4-(1-morpholinyl)pyridine-
at 254 nm.
(1H)-thione (3b)
A mixture of 23.5 g of morpholinium thiocyanate (0.16 mol)
3
and 32.1 g of mesityloxide (0.321 mol) in 125 cm bro-
General procedure for the preparation of 4-
(alkylamino)- and 4-(dialkylamino)-5,6-dihydro-6,6-
dimethylpyridin-2(1H)-thiones 3a–3d
mobenzene gave a product which crystallized over night at
room temperature. It was sucked of, washed with 2-propanol,
and recrystallized from 2-propanol/CH Cl giving 27.1 g
2
2
A mixture of the thiocyanates of the secondary amines,
mesityloxide, and bromobenzene was refluxed over a water
separator, filled with molecular sieves (0.4 nm) as long as
the boiling point of pure bromobenzene was reached. This
process took usually 4 h. The product precipitated from the
solution and was further purified.
(74 %) of 3b as yellowish needles. M.p.: 256 °C, corre-
sponds well with the reported melting point: 255–258 °C [7].
5
2
,6-Dihydro-6,6-dimethyl-4-(1-pyrrolidinyl)pyridine-
(1H)-thione (3c)
A mixture of 54.6 g of pyrrolidinium thiocyanate (0.45 mol)
3
and 90 g of mesityloxide (0.9 mol) in 350 cm bromoben-
4
-(Dimethylamino)-5,6-dihydro-6,6-dimethyl-2(1H)-
zene gave a solution from which some of the solvent was
removed by evaporation in vacuo. The resulting solution
was left in the refrigerator and the product crystallized
overnight. It was sucked of, washed with 2-propanol, and
recrystallized from 2-propanol giving 19 g (20 %) of 3c as
green needles. M.p.: 250 °C, corresponds well with the
reported melting point: 254 °C [12].
pyridinthione (3a)
A mixture of 52 g of N,N-dimethylammonium thiocyanate
3
(
0.5 mol) and 98 g of mesityloxide (1 mol) in 350 cm
bromobenzene reacted to a product which crystallized upon
cooling. It was sucked off, washed with 2-propanol, and
recrystallized from 2-propanol giving 80.6 g (88 %) of 3a
1
23

Synthesis of new tetrahydropyridinylidene ammonium salts and their antiprotozoal potency
Table 1 Antiprotozoal activities (IC50/lM) of compounds 3, 6, and
5,6-Dihydro-6,6-dimethyl-4-(1-piperidinyl)pyridine-
(1H)-thione (3d)
A mixture of 32.5 g of piperidinium thiocyanate (0.23 mol)
1
2
2
Entry
P.falc.NF54
T.b.rhod.
Cytotox.
3
and 45 g of mesityloxide (0.45 mol) in 175 cm bro-
3
3
3
3
6
6
6
6
6
6
6
6
6
6
1
a
b
c
d
a
b
c
d
e
f
[271.3
[220.9
120.3
77.10
10.21
[155.2
2.53
266.4
329.6
80.82
27.86
[357.0
310.4
[326.6
249.2
97.62
19.17
173.1
47.27
3.52
310.4
292.5
161.2
140.0
305.9
212.0
192.4
277.6
[299.2
193.7
[324.5
185.6
80.77
125.0
199.8
188.5
7.78
mobenzene gave a product which crystallized overnight. It
was sucked off, washed with 2-propanol, and recrystallized
from 2-propanol giving 27.4 g (54 %) of 3d as bright
yellow needles. M.p.: 217 °C, corresponds well with the
reported melting point: 217 °C [12].
General procedure for the preparation of 4-
(alkylamino)- and 4-(dialkylamino)-5,6-dihydro-6,6-
dimethylpyridine-2(1H)-thiones 3e, 3g–3j
21.61
7.69
69.93
25.37
68.58
6.99
g
h
i
Compound 7 was dissolved in benzene, glacial acetic acid
was added, and a solution of the primary or secondary
amine in benzene was added too. The mixture was refluxed
for 4 h on a water separator and cooled to room tempera-
ture. The product crystallized from the reaction mixture,
was sucked off, and recrystallized from ethanol or
2-propanol.
j
26.06
0.36
5.40
2
37.35
chl
0.006
mel
mef
0.004
11.37
4-(Azepan-1-yl)-6,6-dimethyl-5,6-dihydropyridine-2(1H)-
Values represent the average of four determinations (two determi-
nations of two independent experiments)
thione (3e, C H N S)
1
3 22 2
3
3
Reaction of 3.15 g of 7 (20 mmol) dissolved in 50 cm
with 1.98 g azepane (20 mmol) dissolved in 16 cm
chl chloroquine, mel melarsoprol, mef mefloquine
Scheme 2
123

W. Seebacher et al.
3
benzene in the presence of 1.2 cm glacial acetic acid
27.10 (2 CH ), 39.89 (C-5), 51.90 (CHCH ), 52.37 (C-6),
3
3
yielded 2.04 g (43 %) of 3e. For analytical purposes, a
further crystallization was done from ethanol after treat-
ment with charcoal. R (CHCl :benzene:EtOH = 4:4:1)
95.47 (C-3), 125.70, 127.04, 128.70 (ArC), 144.43 (ArCq),
150.89 (C-4), 189.21 (C-2) ppm; IR (KBr): vꢀ = 3202,
3041, 2973, 1580, 1543, 1508, 1447, 1415, 1181, 1101,
f
1
3
-1
967, 700 cm ; UV (CH OH): k (log e) = 331 (4.456),
=
0.44; m.p.: 233 °C; H NMR (400 MHz, DMSO-d ):
6
3
0
?
?
d = 1.18 (s, 6H, 2 CH ), 1.47 (br, s, 4H, H-3 ), 1.63 (br, s,
209 (4.166) nm; HRMS (EI ): calcd. C H N S [M ]
15 20 2
3
0
0
4
H, H-2 ), 2.40 (s, 2H, H-5), 3.38 (br, s, 4H, H-1 ), 5.19 (s,
260.1347, found 260.1340.
1
H, H-3), 8.15 (s, 1H, H-1) ppm; C NMR (100 MHz,
3
1
0
0
DMSO-d ): d = 25.52 (C-2 ), 26.11 (C-3 ), 26.94 (2 CH ),
3
4-(Dibenzylamino)-6,6-dimethyl-5,6-dihydropyridine-
2(1H)-thione (3i, C21 S)
Reaction of 6.29 g of 7 (40 mmol) dissolved in 120 cm
benzene with 7.89 g dibenzylamine (40 mmol) dissolved
6
3
0
6.90 (C-5), 49.66 (C-1 ), 52.30 (C-6), 95.64 (C-3), 152.61
H N
24 2
3
(
C-4), 188.40 (C-2) ppm; IR (KBr): vꢀ = 3182, 2968, 2931,
1
1
560, 1542, 1507, 1485, 1449, 1412, 1376, 1363, 1345,
-
164, 1105, 1028, 772 cm ; UV (CH OH): k (log
e) = 346 (4.495), 206 (4.066) nm; HRMS (EI ): calcd.
C H N S [M ] 238.1504, found 238.1516.
1
3
3
in 30 cm benzene in the presence of 2.4 cm glacial acetic
acid yielded 2.24 g (17 %) of 3i. For analytical purposes, a
further crystallization from ethanol was done after treat-
ment with charcoal. R (CHCl :benzene:EtOH = 4:4:1)
3
?
?
1
3 22 2
f
3
6
,6-Dimethyl-4-(phenylamino)-5,6-dihydropyridine-2(1H)-
1
=
0.57; m.p.: 175 °C; H NMR (400 MHz, DMSO-d ):
6
thione (3f)
d = 1.15 (s, 6H, 2 CH ), 2.49 (s, 2H, H-5), 4.59 (s, 4H,
3
This compound was prepared according a reported proce-
dure [8]. Its melting point m.p.: 230 °C (Ref. [8] 234 °C)
corresponds well with the reported one.
ArCH ), 5.25 (s, 1H, H-3), 7.19-7.38 (m, 10H, ArH), 8.41
2
13
br, s, 1H, NH) ppm; C NMR (100 MHz, DMSO-d6):
(
d = 26.99 (2 CH ), 37.33 (C-5), 52.57 (C-6), 53.12
3
4
2
-(tert-Butylamino)-6,6-dimethyl-5,6-dihydropyridine-
(ArCH
137.36 (ArC
(KBr): vꢀ = 3170, 1555, 1509, 1454, 1411, 1373, 1342,
2
), 97.92 (C-3), 126.46, 127.33, 128.87 (ArC),
(1H)-thione (3g, C H N S)
1
q
), 152.66 (C-4), 188.90 (C-2) ppm; IR
1 20 2
3
Reaction of 4.71 g of 7 (30 mmol) dissolved in 100 cm
benzene with 2.19 g tert-butylamine (30 mmol) dissolved
-
1
1178, 1110, 953, 740, 693 cm ; UV (CH
OH): k (log
3
3
3
?
in 20 cm benzene in the presence of 1.7 cm glacial acetic
acid yielded 960 mg (15 %) of 3g after recrystallization
from ethanol. R (CHCl :benzene:EtOH = 4:4:1) = 0.30;
e) = 340 (4.484), 207 (4.375) nm; HRMS (EI ): calcd.
?
S [M ] 336.1660, found 336.1653.
C
21
H
N
2
24
f
3
6,6-Dimethyl-4-(4-chlorophenylamino)-5,6-
1
m.p.: 296 °C; H NMR (400 MHz, DMSO-d ): d = 1.14
6
dihydropyridine-2(1H)-thione (3j)
This compound was prepared according a reported proce-
(
s, 6H, 2 CH ), 1.28 (s, 9H, C(CH ) ), 2.18 (s, 2H, H-5),
3 3 3
5
.27 (s, 1H, H-3), 6.44 (s, 1H, NH), 8.20 (s, 1H, H-1) ppm;
dure [8]. Its melting point m.p.: 245 °C (Ref. [8]
1
3
C NMR (100 MHz, DMSO-d ): d = 26.80 (2 CH ),
6
3
245–247 °C) corresponds well with the reported one.
2
9
8.69 (C(CH ) ), 40.86 (C-5), 51.49, 52.19 (C-6, C(CH ) ),
3 3 3 3
6.31 (C-3), 150.07 (C-4), 188.71 (C-2) ppm; IR (KBr):
General procedure for the preparation of (2,2-
dimethyl-1,2,3,4-tetrahydropyridin-4-
ylidene)ammonium iodides 6a–6j and 12
vꢀ = 3262, 3174, 2969, 1578, 1536, 1510, 1462, 1406,
-
1
366, 1209, 1142, 1105, 1003, 806, 723 cm ; UV
1
(
(
CH OH): k (log e) = 331 (4.351), 210 (3.859) nm; HRMS
3
?
EI ): calcd. C H N S [M ] 212.1347, found 212.1349.
?
1
1 20 2
A mixture of chloroform and methyl iodide was added to
the dihydropyridinethiones 3a–3j or 11 and stirred over
night at room temperature. The solution was concentrated,
cooled with an ice bath, and the methylthio compounds
precipitated upon the addition of ethyl acetate.
6
2
,6-Dimethyl-4-(1-phenylethylamino)-5,6-dihydropyridine-
(1H)-thione (3h, C H N S)
1
5 20 2
3
Reaction of 3.15 g of 7 (20 mmol) dissolved in 60 cm
benzene with 2.42 g of 1-phenylethylamine (20 mmol)
3
dissolved in 15 cm benzene in the presence of 1.2 cm
3
Powdered nickel/aluminum alloy was given into a big
beaker, water was added and solid NaOH was added cau-
tiously. After the main reaction ceased, the beaker was put
into a water bath at 70 °C for 30 min. After that, the liquid
was decanted and the solid nickel was washed with water
until the solution reacted neutral. After that it was washed
twice with ethanol and given to a solution of the methylthio
compound in ethanol. Then it was stirred for 30–45 min at
room temperature. The catalyst was removed by suction
and washed with ethanol. The filtrate and washings were
glacial acetic acid yielded 4.51 g (87 %) of 3h. For
analytical purposes, a further crystallization was done after
treatment with charcoal from 2-propanol. R (CHCl :ben-
f
3
1
zene:EtOH = 4:4:1) = 0.32; m.p.: 208 °C; H NMR
(
400 MHz, DMSO-d ): d = 1.12 (s, 3H, CH ), 1.18 (s,
6
3
3
H, CH ), 1.37 (d, J = 7.0 Hz, 3H, CHCH ), 2.29 (s, 2H,
3 3
H-5), 4.49 (t, J = 6.6 Hz, 1H, CHCH ), 4.91 (s, 1H, H-3),
3
1
3
7
.20–7.35 (m, 6H, NH, ArH), 8.19 (s, 1H, H-1) ppm;
C
NMR (100 MHz, DMSO-d ): d = 23.96 (CHCH ), 26.71,
6
3
1
23

Synthesis of new tetrahydropyridinylidene ammonium salts and their antiprotozoal potency
combined and evaporated in vacuo. The residue was dis-
solved in chloroform, filtered, and the solvent evaporated
in vacuo giving the reduction products as green resins,
which were dissolved in chloroform. Ethyl acetate was
added. The product precipitated, was filtered with suction
and dissolved in chloroform. The solution was treated with
charcoal, filtered, and ethyl acetate was added. The pre-
cipitate was filtered with suction and dried at 100 °C under
reduced pressure.
put at the external bisector of the C–C–C angle at a C–H
˚
distance of 0.95 A and one common isotropic displacement
parameter was refined for these H atoms. The H atoms of
the methyl groups were refined with common isotropic
displacement parameters for the H atoms of the same group
and idealized geometries with tetrahedral angles, enabling
˚
rotation around the X–C bond, and C–H distances of 0.98 A.
For 123 parameters final R indices of R1 = 0.0181 and
2
wR = 0.0469 (GOF = 1.084) were obtained. The largest
-
3
˚
peak in a difference Fourier map was 0.528 e A . The final
atomic parameters, as well as bond lengths and angles are
deposited at the Cambridge Crystallographic Data Centre
N,N-Dimethyl-2,2-dimethyl-1,2,3,4-tetrahydropyridin-4-
iminium iodide (6a, C H IN )
9
17
2
3
A solution of 33.9 g 3a (0.19 mol) in 90 cm of chloroform
reacted with 31.2 g methyl iodide (0.22 mol) dissolved in
(CCDC 1047057).
3
0 cm of chloroform to a precipitate which was treated in
9
N-(2,2-Dimethyl-1,2,3,4-tetrahydropyridin-4-ylidene)-
3
a total volume of 500 cm ethanol with Raney nickel
prepared from 150 g nickel/aluminum alloy and 300 g of
caustic soda giving 31.7 g (74 %) 6a as yellow needles. Rf
morpholinium iodide (6b, C H IN O)
19
1
1
2
3
A solution of 5.8 g 3b (25 mmol) in 10 cm of chloroform
reacted with 4.3 g methyl iodide (31 mmol) dissolved in
3
10 cm of chloroform to precipitate which was were
3
treated in a total volume of 150 cm of ethanol with Raney
(
CHCl :benzene:MeOH:Me NH = 4:4:1:0.5) = 0.09;
3
2
1
m.p.: 148 °C; H NMR (400 MHz, DMSO-d ): d = 1.26
(
6
s, 6H, 2 CH ), 2.85 (s, 2H, H-3), 3.17, 3.24 (2 s, 6H,
nickel prepared from 19 g nickel/aluminum alloy and 38 g
of caustic soda giving 2.8 g (46 %) 6b as yellow needles.
Rf (CHCl :benzene:MeOH:Me NH = 4:4:1:0.5) = 0.11;
3
N(CH ) ), 5.20 (d, J = 6.6 Hz, 1H, H-5), 7.61 (d,
3
2
1
3
J = 6.6 Hz, 1H, H-6), 9.35 (br, s, 1H, H-1) ppm;
C
3
2
1
NMR (100 MHz, DMSO-d ): d = 25.66 (2 CH ), 37.45
m.p.: 138 °C; H NMR (400 MHz, DMSO-d ): d = 1.27
6
3
6
(
C-3), 40.66, 41.18 (N(CH ) ), 51.40 (C-2), 86.35 (C-5),
3
(s, 6H, 2 CH ), 2.86 (s, 2H, H-3), 3.69 (s, br, 8H, N(CH ) ,
2
3
2 2
1
3
1
54.05 (C-6), 165.61 (C-4) ppm; IR (KBr): vꢀ = 3156,
126, 3044, 2955, 1612, 1556, 1420, 1410, 1354, 1278,
O(CH ) ), 5.40 (d, J = 6.2 Hz, 1H, H-5), 7.69 (d,
2 2
1
3
J = 6.2 Hz, 1H, H-6), 9.55 (br, s, 1H, H-1) ppm;
C
-
1
250, 1130, 785 cm ; UV (CH OH): k (log e) = 340
NMR (100 MHz, DMSO-d ): d = 25.69 (2 CH ), 36.84
3
6
3
(
[
4.246), 220 (4.175) nm; HRMS (MALDI): calcd. C H N
17
(C-3), 48.08 (N(CH ) ), 51.56 (C-2), 65.74, 66.36
2 2
9
2
?
MH ] 153.1392, found 153.1375.
(O(CH ) ), 86.32 (C-5), 155.01 (C-6), 164.22 (C-4) ppm;
2
2
IR (KBr): vꢀ = 3134, 3091, 3009, 2961, 2933, 1603, 1552,
1
1
427, 1398, 1368, 1348, 1319, 1258, 1181, 1108, 1066,
-
Crystal structure analysis of 6a
1
036, 951, 741 cm ; UV (CH OH): k (log e) = 343
3
(
4.246), 223 (3.956) nm; HRMS (MALDI): calcd.
All the measurements were performed on a Bruker APEX-II
CCD diffractometer using graphite-monochromatized Mo
?
C H N O [M-I] 195.1497, found 195.1489; calc. C22-
1
1 19 2
?
H N IO [2M-I] 517.2040, found 517.2068.
3
8
4
2
Ka radiation at 100 K on
a
single crystal
(
0.24 9 0.24 9 0.15 mm): C H IN ,
2
M = 280.15,
r
N-(2,2-Dimethyl-1,2,3,4-tetrahydropyridin-4-
9
17
˚
monoclinic, space group P 2 /c, a = 6.5347(4) A,
1
ylidene)pyrrolidinium iodide (6c, C H IN )
11 19
2
˚
˚
3
A solution of 26.4 g 3c (124 mmol) in 40 cm of
chloroform reacted with 21.1 g methyl iodide (148 mmol)
b = 15.0191(8) A, c = 12.1232(8) A, b = 103.884(2)°,
3
V = 1155.07(12) A , Z = 4, dcalc = 1.611 g cm
-3
˚
,
-
1
3
dissolved in 40 cm of chloroform to a precipitate which
3
was treated in a total volume of 300 cm with Raney nickel
l = 2.730 mm . A total of 8007 reflections were collected
(
Hmax = 30.0°), from which 3342 were unique
Rint = 0.0205), with 2957 having I [ 2r(I). The structure
was solved by direct methods (SHELXS-97) [13] and
(
prepared from 100 g nickel/aluminum alloy and 200 g of
caustic soda giving 23 g (73 %) 6c as yellow needles. Rf
(CHCl :benzene:MeOH:Me NH = 4:4:1:0.5) = 0.17;
2
refined by full-matrix least-squares techniques against F
3
2
1
(
SHELXL-97) [13]. The non-hydrogen atoms were refined
m.p.: 192 °C; H NMR (400 MHz, DMSO-d ): d = 1.27
6
with anisotropic displacement parameters without any
constraints. The H atom bonded to N1 was refined without
any positional constraints with an individual isotropic dis-
(s, 6H, 2 CH ), 1.91–1.97 (m, 4H, (CH ) ), 2.84 (s, 2H, H-
3
2 2
3), 3.49 (t, J = 6.2 Hz, 2H, NCH ), 3.66 (t, J = 6.2 Hz,
2
2H, NCH ), 5.09 (d, J = 6.6 Hz, 1H, H-5), 7.58 (d,
2
1
J = 6.6 Hz, 1H, H-6), 9.27 (br, s, 1H, H-1) ppm; C NMR
3
placement parameter. The H atoms of the CH group were
2
refined with a common isotropic displacement parameter
and idealized geometry with approximately tetrahedral angles
(100 MHz, DMSO-d ): d = 24.29, 24.54 ((CH ) ), 25.78
(2 CH ), 38.70 (C-3), 49.52, 49.85 (N(CH ) ), 51.31 (C-2),
3 2 2
6
2 2
˚
and C–H distances of 0.99 A. The H atoms H5 and H6 were
86.95 (C-5), 153.87 (C-6), 162.54 (C-4) ppm; IR (KBr):
123

W. Seebacher et al.
3
7 cm of chloroform to a precipitate which was treated in a
3
total volume of 40 cm ethanol with Raney nickel prepared
vꢀ = 3149, 3119, 3027, 2944, 2867, 1606, 1555, 1532,
-
1
1
428, 1411, 1334, 1287, 1268, 1183, 1136, 760, 730 cm
;
UV (CH OH): k (log e) = 341 (4.303), 220 (4.188) nm;
from 6 g nickel/aluminum alloy and 12 g of caustic soda
giving 668 mg (67 %) 6f as a light yellow crystals. Rf
(CHCl :benzene:MeOH:Me NH = 4:4:1:0.5) = 0.11;
3
?
HRMS (MALDI): calcd. C H N [M-I] 179.1548,
1
1 19 2
found 179.1535.
3
2
1
m.p.: 192 °C; H NMR (400 MHz, DMSO-d ): d = 1.35
6
N-(2,2-Dimethyl-1,2,3,4-tetrahydropyridin-4-
(
s, 6H, 2 CH ), 2.86 (s, 2H, H-3), 5.33 (d, J = 6.2 Hz, 1H,
3
ylidene)piperidinium iodide (6d, C H IN )
2
1
2
21
H-5), 7.38-7.56 (m, 5H, ArH), 7.78 (d, J = 6.2 Hz, 1H,
3
A solution of 12.7 g 3d (57 mmol) in 35 cm of chloro-
form reacted with 9.6 g methyl iodide (68 mmol) dissolved
*
H-6), 10.04 (br, s, 1H, H -1), 10.87 (br, s, 1H, NH ) ppm;
*
1
3
C NMR (100 MHz, DMSO-d ): d = 25.41 (2 CH ),
3
in 35 cm of chloroform to a precipitate which was treated
6
3
3
1
9.65 (C-3), 52.19 (C-2), 85.75 (C-5), 124.42, 127.55,
3
in a total volume of 200 cm ethanol with Raney nickel
29.86 (ArC), 136.89 (ArC ), 157.52 (C-6), 162.81 (C-4)
q
prepared from 55 g nickel/aluminum alloy and 110 g of
ppm; IR (KBr): vꢀ = 3159, 3101, 2999, 1600, 1559, 1538,
caustic soda giving 10.9 g (62 %) 6d as yellow needles. Rf
(
1
7
509, 1492, 1464, 1449, 1372, 1318, 1286, 1265, 1214,
-1
CHCl :benzene:MeOH:Me NH = 4:4:1:0.5) = 0.13;
3 2
57 cm ; UV (CH OH): k (log e) = 219 (4.219), 350
1
3
m.p.: 133 °C; H NMR (400 MHz, DMSO-d ): d = 1.26
6
?
4.180) nm; HRMS (EI ): calcd. C H N [M-HI]
?
(
13 16 2
(
s, 6H, 2 CH ), 1.60 (s, br, 6H, 3CH ), 2.85 (s, 2H, H-3),
3 2
200.1313, found 200.1303.
3
.66 (s, br, 4H, N(CH ) ), 5.38 (d, J = 6.2 Hz, 1H, H-5),
2 2
7
.60 (d, J = 5.5 Hz, 1H, H-6), 9.32 (br, s, 1H, H-1) ppm;
C NMR (100 MHz, DMSO-d ): d = 23.41 (CH ), 25.57
2 CH ), 25.85, 26.97 (2 CH ), 36.98 (C-3), 49.03, 49.23
N-tert-Butyl-N-(2,2-dimethyl-1,2,3,4-tetrahydropyridin-4-
1
3
ylidene)ammonium iodide (6g, C H IN )
11 21
6
2
2
3
A solution of 0.73 g of 3g (3.4 mmol) in 5 cm of
chloroform reacted with 0.55 g methyl iodide (3.9 mmol)
(
(
(
3
2
N(CH ) ), 51.45 (C-2), 86.07 (C-5), 154.29 (C-6), 163.61
2
2
3
dissolved in 5 cm of chloroform to a precipitate which
C-4) ppm; IR (KBr): vꢀ = 3161, 3034, 2939, 2859, 1602,
3
was treated in a total volume of 45 cm ethanol with Raney
1
1
548, 1473, 1453, 1432, 1355, 1340, 1289, 1255, 1184,
-
141, 1015, 784 cm ; UV (CH OH): k (log e) = 343
1
nickel prepared from 6 g nickel/aluminum alloy and 12 g
of caustic soda giving 602 mg (64 %) 6g as light yellow
crystals. R (CHCl :benzene:MeOH:Me NH = 4:4:1:0.5)
3
(
4.314), 220 (4.172) nm; HRMS (MALDI): calcd.
?
C H N [M-I] 193.1705, found 193.1680.
1
2
21
2
f
3
2
1
=
0.14; m.p.: 170 °C; H NMR (400 MHz, DMSO-d ):
6
N-(2,2-Dimethyl-1,2,3,4-tetrahydropyridin-4-yliden)-
d = 1.23 (s, 6H, 2 CH ), 1.37 (s, 9H, C(CH ) ) 2.64 (s, 2H,
3
3 3
azepanium iodide (6e, C H IN )
1
3
23
2
H-3), 5.35 (d, J = 6.2 Hz, 1H, H-5), 7.63 (d, J = 6.2 Hz,
3
A solution of 1.12 g of 3e (4.7 mmol) in 8 cm of chloroform
reacted with 0.76 g methyl iodide (5.4 mmol) dissolved in
1
3
H, H-6), 9.20 (br, s, 2H, H-1, NH) ppm; C NMR
1
(100 MHz, DMSO-d6): d = 25.21 (2 CH3), 28.40
3
cm of chloroform to a precipitate which was treated in a
8
(
C(CH ) ), 40.57 (C-3), 51.77 (C-2), 54.30 (C(CH ) ),
3 3
3
3 3
total volume of 40 cm ethanol with Raney nickel prepared
from 6 g nickel/aluminum alloy and 12 g of caustic soda
giving 755 mg 6e (74 %) as light yellow crystals. Rf
86.04 (C-5), 155.08 (C-6), 163.05 (C-4) ppm; IR (KBr):
vꢀ = 3216, 3169, 3113, 3064, 3011, 2976, 2937, 1613,
1
1
565, 1543, 1514, 1467, 1420, 1381, 1365, 1347, 1281,
-1
(
CHCl :benzene:MeOH:Me NH = 4:4:1:0.5) = 0.17; m.p.:
3 2
268, 1203, 1184, 1139, 802, 716 cm ; UV (CH OH): k
1
3
1
41 °C; H NMR (400 MHz, DMSO-d ): d = 1.27 (s, 6H, 2
3
6
?
(
log e) = 334 (4.322), 219 (4.214) nm; HRMS (EI ):
0
0
CH ), 1.50 (s, 4H, H-3 ), 1.67–1.72 (m, 4H, H-2 ), 2.88 (s, 2H,
0
?
calcd. C H N [M-HI] 180.1626, found 180.1633.
11 20 2
H-3), 3.68-3.73 (m, 4H, H-1 ), 5.27 (d, J = 6.6 Hz, 1H, H-5),
1
3
C
7
.60 (d, J = 6.6 Hz, 1H, H-6), 9.39 (br, s, 1H, H-1) ppm;
0
N-2,2-Dimethyl-N-(1-phenylethyl)-1,2,3,4-
NMR (100 MHz, DMSO-d ): d = 25.22 (C-2 ), 25.36 (2
tetrahydropyridin-4-iminium iodide (6h, C H IN )
15 21
6
2
0
0
3
A solution of 1.48 g of 3h (5.7 mmol) in 8 cm of
chloroform reacted with 1.04 g methyl iodide (7.3 mmol)
CH ), 25.44, 25.66 (C-3 ), 28.14 (C-2 ), 36.86 (C-3), 51.12
3
0
0
(
C-1 ), 51.53 (C-2), 51.69 (C-1 ), 86.03 (C-5), 154.28 (C-6),
3
dissolved in 8 cm of chloroform to a precipitate which
3
was treated in a total volume of 45 cm ethanol with Raney
1
2
1
64.94 (C-4) ppm; IR (KBr): vꢀ = 3160, 3036, 2965, 2931,
862, 1601, 1554, 1484, 1465, 1451, 1427, 1369, 1343, 1277,
-1
249, 1187, 1174, 785 cm ; UV (CH OH): k (log e) = 342
nickel prepared from 6 g nickel/aluminum alloy and 12 g
of caustic soda giving 710 mg (65 %) 6h as light yellow
crystals. R (CHCl :benzene:MeOH:Me NH = 4:4:1:0.5)
3
?
(
4.355), 220 (4.274) nm; HRMS (EI ): calcd. C H N [M-
13 22 2
?
HI] 206.1783, found 206.1792.
f
3
2
1
=
0.17; m.p.: 167 °C; H NMR (400 MHz, DMSO-d ):
6
2
,2-Dimethyl-N-phenyl-1,2,3,4-tetrahydropyridin-4-
d = 1.20 (s, 3H, CH
J = 6.6 Hz, 3H, CHCH
J = 7.0 Hz, 1H, CHCH ), 5.09 (d, J = 6.2 Hz, 1H, H-5),
7.29-7.39 (m, 5H, ArH), 7.62 (d, J = 6.2 Hz, 1H, H-6),
3
), 1.26 (s, 3H, CH
3
), 1.49 (d,
iminium iodide (6f, C H IN )
2
3
), 2.70 (s, 2H, H-3), 4.91 (q,
1
3
17
3
A solution of 0.73 g 3f (3.1 mmol) in 7 cm of chloroform
reacted with 0.88 g methyl iodide (6.2 mmol) dissolved in
3
1
23

Synthesis of new tetrahydropyridinylidene ammonium salts and their antiprotozoal potency
1
.63 (br, s, 2H, H-1, NH) ppm; C NMR (100 MHz,
3
?
?
9
(EI ): calcd. C H ClN [M-HI] 234.0924, found
13 15 2
DMSO-d ): d = 22.61 (CHCH ), 25.25, 25.34 (2 CH ),
234.0926.
6
3
3
3
1
6
2
1
9.70 (C-3), 51.86 (C-2), 53.12 (CHCH ), 85.02 (C-5),
3
6
,6-Dimethyl-2-thioxo-4-piperidone (7)
26.01, 127.69, 128.91 (ArC), 142.33 (ArC ), 155.97 (C-
q
This compound was prepared according a reported proce-
dure [9]. Its melting point m.p.: 139 °C (Ref. [9] 138 °C)
corresponds well with the reported one.
), 163.76 (C-4) ppm; IR (KBr): vꢀ = 3169, 3027, 2996,
892, 1605, 1572, 1542, 1507, 1453, 1418, 1383, 1372,
-
1
;
344, 1296, 1272, 1185, 1177, 1124, 783, 734, 697 cm
UV (CH OH): k (log e) = 336 (4.259), 218 (4.166) nm;
5,6-Dihydro-6,6-dimethyl-4-(1-pyrrolidinyl)-thiopyrane-
2(1H)-thione (8)
3
?
?
HRMS (EI ): calcd. C H N [M-HI] 228.1626, found
1
5 20 2
2
28.1643.
This compound was prepared according a reported proce-
dure [14]. Its melting point m.p.: 230 °C (Ref. [14] 233 °C)
corresponds well with the reported one.
N,N-Dibenzyl-2,2-dimethyl-1,2,3,4-tetrahydropyridin-4-
iminium iodide (6i, C H IN )
25
2
1
2
3
A solution of 1.22 g of 3i (3.6 mmol) in 6 cm of
chloroform reacted with 0.67 g methyl iodide (4.7 mmol)
N-[2,3-Dihydro-2,2-dimethyl-6-(methylthio)-4H-
thiopyran-4-ylidene]pyrrolidinium iodide (9)
3
dissolved in 6 cm of chloroform to a precipitate which
This compound was prepared according a reported proce-
dure [15]. Its melting point m.p.: 195 °C (Ref. [15] 198 °C)
corresponds well with the reported one.
3
was treated in a total volume of 40 cm ethanol with Raney
nickel prepared from 6 g nickel/aluminum alloy and 12 g
of caustic soda giving 243 mg (25 %) 6i as a light yellow
crystals. R (CHCl :benzene:MeOH:Me NH = 4:4:1:0.5)
N-[2,3-Dihydro-2,2-dimethyl-6-(phenylimino)-4H-
thiopyran-4-ylidene]pyrrolidinium iodide (10)
This compound was prepared according a reported proce-
dure [16]. Its melting point m.p.: 264 °C (Ref. [16] 262 °C)
corresponds well with the reported one.
f
3
2
1
=
0.24; m.p.: 135 °C; H NMR (400 MHz, DMSO-d ):
6
d = 1.26 (s, 6H, 2 CH ), 3.01 (s, 2H, H-3), 4.87–4.93 (m,
3
4
H, 2 NCH ), 5.34 (d, J = 6.2 Hz, 1H, H-5), 7.25–7.40 (m,
2
1
0H, ArH), 7.71 (d, J = 5.5 Hz, 1H, H-6), 9.83 (br, s, 1H,
1
3
H-1) ppm; C NMR (100 MHz, DMSO-d ): d = 25.36 (2
5,6-Dihydro-6,6-dimethyl-1-phenyl-4-(1-pyrrolidinyl)-
6
CH ), 37.41 (C-3), 51.92 (C-2), 54.42 (2 NCH ), 87.57 (C-
2
pyridine-2(1H)-thione (11)
A suspension of 8 g of 10 (19 mmol) in 140 cm of 1 N
3
3
5
1
3
1
), 126.82, 128.00, 129.06 (ArC), 134.43, 136.05 (ArCq),
55.85 (C-6), 166.08 (C-4) ppm; IR (KBr): vꢀ = 3145,
102, 3024, 2946, 1547, 1496, 1451, 1413, 1346, 1325,
NaOH was stirred for 30 min. It was extracted 3 times with
chloroform, the combined organic phases were dried over
sodium sulfate, filtered and the solvent evaporated in
vacuo. The residue was dried by threefold codistillation
-
1
285, 1255, 1178, 755, 739, 706 cm ; UV (CH OH): k
3
?
(log e) = 211 (4.419), 346 (4.388) nm; HRMS (EI ):
calcd. C H N [M-HI] 304.1939, found 304.1952.
?
3
with benzene and then 35 cm DMF were added. The
2
1 24 2
solution was refluxed overnight and then evaporated. The
residue was recrystallized from ethyl acetate giving 2.76 g
N-(4-Chlorophenyl)-2,2-dimethyl-1,2,3,4-
tetrahydropyridin-4-iminium iodide (6j, C H ClIN )
2
1
3
16
(
51 %) of 10. Its melting point m.p.: 235 °C (Ref. [17]
34 °C) corresponds well with the reported one.
3
A solution of 2.14 g of 3j (8.7 mmol) in 10 cm of
chloroform reacted with 1.5 g methyl iodide (10.5 mmol)
2
3
dissolved in 10 cm of chloroform to a precipitate which
N-[2,3-Dihydro-2,2-dimethyl-1-phenyl-4H-pyridin-4-yli-
3
was treated in a total volume of 40 cm ethanol with Raney
dene]pyrrolidinium iodide (12, C H IN )
1
7
23
2
3
A solution of 0.8 g of 11 (2.77 mmol) in 5 cm of
chloroform reacted with 0.47 g methyl iodide (3.35 mmol)
nickel prepared from 6 g nickel/aluminum alloy and 12 g
of caustic soda giving 770 mg (69 %) 6j as light yellow
crystals. R (CHCl :benzene:MeOH:Me NH = 4:4:1:0.5)
3
dissolved in 5 cm of chloroform to a precipitate which
3
was treated in a total volume of 50 cm ethanol with Raney
f
3
2
1
=
0.17; m.p.: 174 °C; H NMR (400 MHz, DMSO-d ):
6
d = 1.35 (s, 6H, 2 CH ), 2.85 (s, 2H, H-3), 5.35 (d,
nickel prepared from 6 g nickel/aluminum alloy and 12 g
of caustic soda giving 155 mg (17 %) 12 as light yellow
precipitate after additional crystallization from acetone. Rf
(CHCl :benzene:MeOH:Me N = 4:4:1:0.5) = 0.39; m.p.:
3
J = 5.9 Hz, 1H, H-5), 7.34–7.57 (m, 4H, ArH), 7.82 (d,
*
J = 5.9 Hz, 1H, H-6), 10.11 (br, s, 1H, H -1), 10.97 (br, s,
*
H, NH ) ppm;
13
1
C NMR (100 MHz, DMSO-d6):
3
3
1
d = 24.39 (2 CH ), 38.56 (C-3), 51.26 (C-2), 84.97 (C-
175 °C; H NMR (400 MHz, DMSO-d ): d = 1.30 (s, 6H,
3
6
5
), 125.25, 128.86 (ArC), 130.60, 134.63 (ArC ), 156.90
q
2 CH ), 1.97–2.03 (m, 4H, 2 CH ), 3.14 (s, 2H, H-3), 3.62
3
2
(
C-6), 161.58 (C-4) ppm; IR (KBr): vꢀ = 3154, 3095, 2965,
(t, J = 5.9 Hz, 2H, NCH ), 3.78 (t, J = 6.2 Hz, 2H,
2
1
1
597, 1536, 1508, 1487, 1462, 1408, 1371, 1341, 1307,
NCH ), 5.38 (d, J = 7.0 Hz, 1H, H-5), 7.35–7.47 (m, 2H,
2
-
1
280, 1260, 1216, 1178, 1088, 1010, 744 cm ; UV
ArH), 7.49–7.52 (m, 3H, ArH), 7.61 (d, J = 7.0 Hz, 1H,
1
3
(
CH OH): k (log e) = 220 (4.308), 339 (4.194) nm; HRMS
H-6) ppm; C NMR (100 MHz, DMSO-d ): d = 24.32
3
6
123

W. Seebacher et al.
(
CH ), 24.41 (2 CH ), 24.53 (CH ), 40.12 (C-3), 50.24,
2
and the plate incubated at 37 °C under a 5 % CO atmo-
3
2
2
3
sphere for 72 h. Alamar Blue (10 mm ) was then added to
5
1
0.30 (2 NCH ), 58.15 (C-2), 90.01 (C-5), 127.97, 128.98,
2
29.63 (ArC), 141.04 (ArC ), 155.81 (C-6), 163.22 (C-4)
q
each well and incubation continued for a further 2–4 h. The
plate was then read with a Millipore Cytofluor 2300 using
an excitation wavelength of 530 nm and emission wave-
length of 590 nm [23]. Fluorescence development was
expressed as percentage of the control, and IC50 values
determined. Melarsoprol served as standard. Cytotoxicity
was assessed using the same assay and L-6 cells using
mefloquine as standard.
ppm; IR (KBr): vꢀ = 2936, 1600, 1548, 1488, 1451, 1373,
1
7
343, 1322, 1305, 1276, 1214, 1179, 1128, 974, 853, 776,
-
1
08 cm ; UV (CH OH): k (log e) = 351 (4.375), 219
3
?
?
(
4.328) nm; HRMS (EI ): calcd. C H N [M-HI]
17 22 2
2
54.1783, found 254.1784.
Antimalarial activity
In vitro activity against erythrocytic stages of P. falciparum
3
was determined using a H-hypoxanthine incorporation
References
assay [18, 19]), using the drug-sensitive NF54 strain
1
2
. WHO (2013) World Malaria report
. Brun R, Blum J, Chappuis F, Burri C (2010) Lancet 375:148
(
Schipol Airport, The Netherlands [20]) or the multiresis-
tant strain K and the standard drug chloroquine (Sigma
1
3. Rashid K, Wachira FN, Nyakriki JN, Isaac AO (2014) Parasitol
Int 63:417
4
C6628). Compounds were dissolved in DMSO at 10 mg/
3
cm and added to parasite cultures incubated in RPMI 1640
. Hawley SR, Bray PG, Mungthin M, Atkinson JD, O’Neill PM,
Ward SA (1998) Antimicrob Agents Chemother 42:682
. Aissaoui H, Boss C, Corminboeuf O, Franz MC, Grisostomi C
(2011) Novel Bi-amides as antimalarial agents. US Patent
2011/0224110 A1, 15 Sept 2011 (2010) Chem Abstr 153:11260
. Ge JF, Arai C, Yang M, Bakar MA, Lu J, Ismail NSM, Wittlin S,
Kaiser M, Brun R, Charman SA, Nguyen T, Morizzi J, Itoh I,
Ihara M (2010) ACS Med Chem Lett 1:360
medium without hypoxanthine, supplemented with HEPES
3
5
3
(
5.94 g/dm ), NaHCO (2.1 g/dm ), neomycin (100
3
3
U/cm ), Albumax (5 g/dm ) and washed human red cells
?
A at 2.5 % haematocrit (0.3 % parasitaemia). Serial drug
R
3
6
7
dilutions of eleven 3-fold dilution steps covering a range
3
from 100 to 0.002 lg/cm were prepared. The 96-well
. Zigeuner G, Schweiger K (1976) Monatsh Chem 107:1361
plates were incubated in a humidified atmosphere at 37 °C;
8. Zigeuner G, Lintschinger BW, Fuchsgruber A, Kollmann K
1976) Monatsh Chem 107:155
. Zigeuner G, Schweiger K, Fuchsgruber A (1981) Monatsh Chem
12:187
0. Johnson CK (1965) ORTEP Report ORNL-3794. Oak Ridge
National Laboratory, Tennessee
1. Zigeuner G, Kollmann K, Lintschinger WB, Fuchsgruber A
3
% CO , 3 % O , 93 % N . After 48 h 50 mm of H-
3
(
4
2 2 2
9
hypoxanthine (=0.5 lCi) was added to each well of the
plate. The plates were incubated for a further 24 h under
1
1
1
1
the same conditions. The plates were then harvested with a
TM
Betaplate
cell harvester (Wallac, Zurich, Switzerland),
(
1976) Monatsh Chem 107:183
2. Schweiger K, Fuchsgruber A, Zigeuner G (1977) Monatsh Chem
08:243
13. Sheldrick M (2008) Acta Cryst A64:112
and the red blood cells transferred onto a glass fiber filter
then washed with distilled water. The dried filters were
1
3
inserted into a plastic foil with 10 cm of scintillation fluid,
TM
1
1
1
1
4. Schweiger K (1980) Monatsh Chem 111:1175
5. Schweiger K (1982) Monatsh Chem 113:1283
6. Schweiger K (1983) Monatsh Chem 114:317
7. Schweiger K (1983) Monatsh Chem 114:581
18. Desjardins RE, Canfield CJ, Haynes JD, Chulay JD (1979)
Antimicrob Agents Chemother 16:710
and counted in a Betaplate
liquid scintillation counter
(
Wallac, Zurich, Switzerland). IC50 values were calculated
from sigmoidal inhibition curves by linear regression [21]
using Microsoft Excel. Chloroquine was used as control.
1
9. Matile H, Pink JRL (1990) Plasmodium falciparum malaria
parasite cultures and their use in immunology. In: Lefkovits I,
Pernis B (eds) Immunological methods. Academic Press, San
Diego, p 221
Antitrypanosomal activity and cytotoxicity
3
Minimum Essential Medium (50 mm ) supplemented
2
0. Ponnudurai T, Leeuwenberg AD, Meuwissen JH (1981) Trop
Geogr Med 33:50
1. Huber W, Koella JC (1993) Acta Trop 55:257
2. Baltz T, Baltz D, Giroud C, Crockett J (1985) EMBO J 4:1273
according to Baltz et al. [22] with 2-mercaptoethanol and
1
5 % heat-inactivated horse serum was added to each well
2
2
of a 96-well microtiter plate. Serial drug dilutions were
3
added to the wells. Then 50 mm of trypanosome suspen-
23. R a¨ z B, Iten M, Grether-B u¨ hler Y, Kaminsky R, Brun R (1997)
Acta Trop 68:139
sion (T.b.rhodesiense STIB 900) was added to each well
1
23